Means for the modulation of processes mediated by retinoid receptors and compounds useful therefor

ABSTRACT

In accordance with the present invention, there are provided methods to modulate processes mediated by retinoid receptors, employing high affinity, high specificity ligands for such receptors. In one aspect of the present invention, there are provided ligands which are more selective for the retinoid X receptor than is retinoic acid (i.e., rexoids). In another aspect of the present invention, alternative ligands (other than retinoic acid) have been discovered which are capable of inducing retinoic acid receptor mediated processes. In yet another aspect, methods have been developed for the preparation of such retinoid receptor ligands from readily available compounds.

FIELD OF THE INVENTION

[0001] The present invention relates to intracellular receptors, andligands therefor. In a particular aspect, the present invention, relatesto methods for modulating processes mediated by retinoid receptors.

BACKGROUND OF THE INVENTION

[0002] A central problem in eukaryotic molecular biology continues to bethe elucidation of molecules and mechanisms that mediate specific generegulation. in response to exogenous inducers such as hormones or growthfactors. As part of the scientific attack on this problem, a great dealof work has been done in efforts to identify exogenous inducers whichare capable of mediating specific gene regulation.

[0003] Although much remains to be learned about the specifics of generegulation, it is known that exogenous inducers modulate genetranscription by acting in concert with intracellular components,including intracellular receptors and discrete DNA sequences known ashormone response elements (HREs).

[0004] As additional members of the steroid/thyroid superfamily. ofreceptors are identified, the search for exogenous inducers for suchnewly discovered receptors (i.e., naturally occurring (or synthetic)inducers) has become an important part of the effort to learn about thespecifics of gene regulation.

[0005] The retinoid members of the steroid/thyroid superfamily ofreceptors, for example, are responsive to compounds referred to asretinoids, which include retinoic acid, retinol (vitamin A), and aseries of natural and synthetic derivatives which have been found toexert profound effects on development and differentiation in a widevariety of systems.

[0006] The identification of compounds which interact with retinoidreceptors, and thereby affect transcription of genes which areresponsive to retinoic acid (or other metabolites of vitamin A), wouldbe of significant value, e.g., for therapeutic applications.

[0007] Recently, a retinoic acid dependent transcription factor,referred to as RAR-alpha (retinoic acid receptor-alpha), has beenidentified. Subsequently, two additional RAR-related genes have beenisolated; thus there are now at least three different RAR subtypes(alpha, beta and gamma) known to exist in mice and humans. Theseretinoic acid receptors (RARs) share homology with the superfamily ofsteroid hormone and thyroid hormone receptors and have been shown toregulate specific gene expression by a similar ligand-dependentmechanism [Umesono et al., Nature 336: 262 (1988)]. These RAR subtypesare expressed in distinct patterns throughout development and in themature organism.

[0008] More recently, additional novel members of the steroid/thyroidsuperfamily of receptors have been identified, such as, for example,retinoid X receptor-alpha [RXR-α; see Mangelsdorf et al., in Nature 345:224-229 (1990)], retinoid X receptor-beta [RXR-β; see Hamada et al.,Proc. Natl. Acad. Sci. USA 86: 8289-8293 (1989)], and retinoid Xreceptor-gamma [RXR-γ; see Mangelsdorf et al., Genes and Development6:329-344 (1992)]. While these novel receptors are responsive toretinoic acid, the primary exogenous inducer(s) for these receptors havenot been identified.

[0009] Although both RAR and RXR respond to retinoic acid in vivo, thereceptors differ in several important aspects. First, RAR and RXR aresignificantly divergent in primary structure (e.g., the ligand bindingdomains of RARα and RXRα have only 27% amino acid identity). Thesestructural differences are reflected in different relative degrees ofresponsiveness of RAR and RXR to various vitamin A metabolites andsynthetic retinoids. In addition, distinctly different patterns oftissue distribution are seen for RAR and RXR. In contrast to the RARs,which are not expressed at high levels in the visceral tissues, RXRαmRNA has been shown to be most abundant in the liver, kidney, lung,muscle and intestine. Finally, response elements have recently beenidentified in the cellular retinol binding protein type II (CRBPII) andapolipoprotein AI genes which confer responsiveness to RXR, but not RAR.Indeed, RAR has also been recently shown to repress RXR-mediatedactivation through the CRBPII RXR response element. These data, inconjunction with the observation that both RAR and RXR can activatethrough the RAR response element of the RARβ promoter, indicate that thetwo retinoic acid responsive pathways are not simply redundant, butinstead manifest a complex interplay.

[0010] In view of the related, but clearly distinct nature of thesereceptors, the identification of ligands which are more selective forthe retinoid X receptor than is retinoic acid would be of great value inselectively controlling processes mediated by one or both of theseretinoid receptor types.

[0011] Other information helpful in the understanding and practice ofthe present invention can be found in commonly assigned, co-pending U.S.patent application Ser. Nos. 108,471, filed Oct. 20, 1987 (now issued asU.S. Pat. No. 5,071,773); 276,536, filed Nov. 30, 1988 (now issued asU.S. Pat. No. 4,981,784); 325,240, filed Mar. 17, 1989; 370,407, filedJun. 22, 1989; and 438,757, filed Nov. 16, 1989, all of which are herebyincorporated herein by reference in their entirety.

BRIEF DESCRIPTION OF THE INVENTION

[0012] In accordance with the present invention, we have developedmethods to modulate retinoid receptor mediated processes, employing highaffinity, high specificity ligands for such receptors.

[0013] In a particular aspect of the present invention, there areprovided ligands which are high affinity, high specificity ligands forretinoid receptors. Thus, in one aspect of the present invention, thereare provided ligands which are more selective for the retinoid Xreceptor than is all-trans-retinoic acid. In another aspect of thepresent invention, we have discovered alternative ligands (other thanall-trans-retinoic acid) which are capable of inducing retinoic acidreceptor mediated processes.

[0014] In yet another aspect of the present invention, we have developedmethods for the preparation of such retinoid receptor ligands fromreadily available retinoid compounds.

BRIEF DESCRIPTION OF THE FIGURES

[0015]FIG. 1 is a transactivation profile of various HPLC fractionsobtained from retinoic acid (RA)-treated S2 cells.

[0016]FIG. 2a is a comparison of the transactivation profile ofall-trans-retinoic acid (RA) on RAR-alpha and RXR-alpha.

[0017]FIG. 2b is a similar comparison to that shown in FIG. 2a,employing HPLC fraction 18 (instead of RA).

[0018]FIG. 3 presents several activation profiles for analysis ofRXR-alpha or RAR-alpha activation by various retinoic acid isomers.Panel a. represents experiments done in insect S2 cells, while panels b.and c. represent experiments done in mammalian CV-1 cells. In thefigure, closed circles are used to designate 9-cis-retinoic acid, opencircles are used for all-trans-retinoic acid, open triangles are usedfor 13-cis-retinoic acid and open squares are used for 11-cis-retinoicacid.

[0019]FIG. 4 presents the results of saturation binding analysis of9-cis-retinoic acid. Cell extracts were incubated with increasingconcentrations of tritiated retinoid in the absence (total binding) orpresence (non-specific binding) of 200-fold excess non-tritiatedretinoid. Non-specific binding was subtracted from total binding andplotted as specific binding. The data shown in FIG. 4a representspecific [³H]-9-cis-retinoic acid binding to RXRα (closed circles) ormock (open circles) extracts; or specific [³−H]-all-trans-retinoic acidbinding to RXRα (open squares).

[0020]FIG. 4b presents a Scatchard analysis, wherein specific9-cis-retinoic acid binding to RXRα in (a) was transformed by Scatchardanalysis and plotted. Linear regression yielded a Kd=11.7 nM (r=0.86).

[0021]FIG. 5 presents a DNA-cellulose column profile of radiolabelled9-cis-retinoic acid bound to baculovirus expressed RXR. In FIG. 5a,sample cell extracts containing RXRα protein were labelled with 10 nM[³H]-9-cis-retinoic acid in the absence (open squares) or presence (opencircles) of 200-fold excess non-radioactive 9-cis-retinoic acid, andthen applied to the DNA-cellulose column. Fall-through radioactivity wasmonitored until a consistent baseline was established. DNA-bindingcomponents were then eluted with a linear salt gradient. The peakradioactive fractions (labelled 1-15) were then subjected to immunoblotanalysis using an hRXRα-specific antisera. The peak radioactive fraction(indicated by an arrow) co-migrated exactly with the peak amount ofRXRα-specific protein.

[0022] In FIG. 5b, the peak radioactive fraction of the DNA-cellulosecolumn is shown to contain 9-cis-retinoic acid. The peak fraction (arrowin (a)) was extracted and analyzed on a C₁₈ column developed with mobilephase G. As shown, 0.95% of the extracted radioactivity co-elutes withauthentic 9-cis-retinoic acid (absorbance peak).

[0023]FIG. 6 is a comparison of the transactivation profile forRXR-alpha in the presence of 9-cis-retinoic acid employing a luciferasereporter containing the retinoid response element derived from eitherthe apolipoprotein A1 gene (APOA13) or cellular retinol binding protein,type II (CRBPII).

DETAILED DESCRIPTION OF THE INVENTION

[0024] In accordance with the present invention, there is provided amethod for modulating process(es) mediated by retinoid receptors, saidmethod comprising conducting said process(es) in the presence of at.least one compound of the structure:

[0025] wherein:

[0026] unsaturation between carbon atoms C⁹ and C¹⁰ has a cisconfiguration, and one or both sites of unsaturation between carbonatoms C¹¹ through C¹⁴ optionally have a cis configuration;

[0027] “Ring” is a cyclic moiety, optionally having one or moresubstituents thereon;

[0028] Z is selected from carboxyl (—COOH), carboxaldehyde (—COH),hydroxyalkyl [—(CR′₂)_(n)—OH, wherein each R′ is independently selectedfrom hydrogen or a lower alkyl and n falls in the range of 1 up to about4], thioalkyl [—(CR′₂)_(n)—SH, wherein R′ and n are as defined above],hydroxyalkyl phosphate [—(CR′₂)_(n)—OP(OM)₃, wherein R′ and n are asdefined above and M is hydrogen, lower alkyl, or a cationic species suchas Na⁺Li⁺, K⁺, and the like], alkyl ether of a hydroxyalkyl group[—(CR′₂)_(n)—OR′, wherein R′ and n are as defined above], alkylthioether of a thioalkyl group [—(CR′₂)_(n)—SR′, wherein R′ and n are asdefined above], esters of hydroxyalkyl groups [—(CR′₂)_(n)—O—CO—R′,wherein R′ and n are as defined above], thioesters of hydroxyalkyl group[—(CR′₂)_(n)—O—CS—R′, wherein R′ and n are as defined above], esters ofthioalkyl groups [—(CR′₂)_(n)—S—CO—R′, wherein R′ and n are as definedabove], thioesters of thioalkyl groups [—(CR′₂)_(n)—S—CS—R′, wherein R′and n are as defined above], aminoalkyl [—(CR′₂)_(n)—NR′₂, wherein R′and n are as defined above], N-acyl aminoalkyl [—(CR′₂)_(n)—NR′—CO—R″,wherein R′ and n are as defined above and R″ is a lower alkyl orbenzyl], carbamate [—(CR′₂)_(n)—NR′—CO—OR′ or —(CR′₂)_(n)—O—CO—NR′₂,wherein R′ and n are as defined above], and the like; and

[0029] each R is independently selected from H, halogen, alkyl, aryl,hydroxy, thiol, alkoxy, thioalkoxy, amino, or any of the Z substituents,and the like; or

[0030] any two or more of the R groups can be linked to one another toform one or more ring structures.

[0031] Exemplary R groups in the latter situation are selected fromalkylene, oxyalkylene, thioalkylene, and the like.

[0032] As employed herein, the term “modulate” refers to the ability ofa ligand for a member of the steroid/thyroid superfamily to induceexpression of gene(s) maintained under hormone expression control, or torepress expression of gene(s) maintained under such control.

[0033] As employed herein, the phrase “processes mediated by retinoidreceptors” refers to biological, physiological, endocrinological, andother bodily processes which are mediated by receptor or receptorcombinations which are responsive to natural or synthetic retinoids, ornatural or synthetic compounds as defined herein (referred to herein as“rexoids” because of the ability of many of the compounds describedherein to selectively activate retinoid X receptors). Modulation of suchprocesses can be accomplished in vitro or in vivo. In vivo modulationcan be carried out in a wide range of subjects, such as, for example,humans, rodents, sheep, pigs, cows, and the like.

[0034] Exemplary receptors which are responsive to retinoids, andnatural or synthetic compounds as defined herein (i.e., “rexoids”),include retinoic acid receptor-alpha, retinoic acid receptor-beta,retinoic acid receptor-gamma, and splicing variants encoded by the genesfor such receptors; retinoid X receptor-alpha, retinoid X receptor-beta,retinoid X receptor-gamma, and splicing variants encoded by the genesfor such receptors; as well as various combinations thereof (i.e.,homodimers, homotrimers, heterodimers, heterotrimers, and the like),including combinations of such receptors with other members of thesteroid/thyroid superfamily of receptors with which the retinoidreceptors may interact by forming heterodimers, heterotrimers, andhigher heteromultimers. For example, the retinoic acid receptor-alphamay form a heterodimer with retinoid X receptor-alpha, the retinoic acidreceptor-beta may form a heterodimer with retinoid X receptor-alpha,retinoic acid receptor-gamma may form a heterodimer with retinoid Xreceptor-alpha, retinoid X receptor-alpha may form a heterodimer withthyroid receptor, retinoid X receptor-beta may form a heterodimer withvitamin D receptor, retinoid X receptor-gamma may form a heterodimerwith retinoic acid receptor-alpha, and the like.

[0035] As employed herein, the phrase “members of the steroid/thyroidsuperfamily of receptors” (also known as “nuclear receptors” or“intracellular receptors”) refers to hormone binding proteins thatoperate as ligand-dependent transcription factors, including identifiedmembers of the steroid/thyroid superfamily of receptors for whichspecific ligands have not yet been identified (referred to hereinafteras “orphan receptors”). These hormone binding proteins have theintrinsic ability to bind to specific DNA sequences. Following binding,the transcriptional activity of target gene (i.e., a gene associatedwith the specific DNA sequence). is modulated as a function of theligand bound to the receptor.

[0036] The DNA-binding domains of all of these nuclear receptors arerelated, consisting of 66-68 amino acid residues, and possessing about20 invariant amino acid residues, including nine cysteines.

[0037] A member of the superfamily can be identified as a protein whichcontains the above-mentioned invariant amino acid residues, which arepart of the DNA-binding domain of such known steroid receptors as thehuman glucocorticoid receptor (amino acids 421-486), the estrogenreceptor (amino acids 185-250), the mineralocorticoid receptor (aminoacids 603-668), the human retinoic acid receptor (amino acids 88-153).The highly conserved amino acids of the DNA-binding domain of members ofthe superfamily are as follows: (SEQ ID No 1) Cys - X - X - Cys - X -X - Asp* - X - Ala* - X - Gly* - X - Tyr* - X - X - X - X - Cys - X -X - Cys - Lys* - X - Phe - Phe - X - Arg* - X - X - X - X - X - X - X -X - X - (X - X -) Cys - X - X - X - X - X - (X - X - X -) Cys - X - X -X - Lys - X - X - Arg - X - X - Cys - X - X -, Cys - Arg* - X - X -Lys* - Cys - X - X - X - Gly* - Met;

[0038] wherein X designates non-conserved amino acids within theDNA-binding domain; the amino acid residues denoted with an asterisk areresidues that are almost universally conserved, but for which variationshave been found in some identified hormone receptors; and the residuesenclosed in parenthesis are optional residues (thus, the DNA-bindingdomain is a minimum of 66 amino acids in length, but can contain severaladditional residues).

[0039] Exemplary members of the steroid/thyroid superfamily of receptorsinclude steroid receptors such as glucocorticoid receptor,mineralocorticoid receptor, progesterone receptor, androgen receptor,vitamin D₃ receptor, and the like; plus retinoid receptors, such asRARα, RARβ, RARγ, and the like, plus RXRα, RXRβ, RXRγ, and the like;thyroid receptors, such as TRα, TRβ, and the like; as well as other geneproducts which, by their structure and properties, are considered to bemembers of, the superfamily, as defined hereinabove. Examples of orphanreceptors include HNF4 [see, for example, Sladek et al., in Genes &Development 4: 2353-2365 (1990)], the COUP family of receptors [see, forexample, Miyajima et al., in Nucleic Acids Research 16: 11057-11074(1988), Wang et al., in Nature 340: 163-166 (1989)], COUP-like receptorsand COUP homologs, such as those described by Mlodzik et al., in Cell60: 211-224 (1990) and Ladias et al., in Science 251: 561-565 (1991),the ultraspiracle receptor [see, for example, Oro et al., in Nature 347:298-301 (1990)], and the like.

[0040] Processes capable of being modulated by retinoid receptors, inaccordance with the present invention, include in vitro cellulardifferentiation and proliferation, in vitro proliferation of melanomacell lines, in vitro differentiation of mouse teratocarcinoma cells (F9cells), in vitro differentiation of human epidermal keratinocytes, limbmorphogenesis, regulation of cellular retinol binding protein (CRBP),and the like. As readily recognized by those of skill in the art, theavailability of ligands for the retinoid X receptor makes it possible,for the first time, to carry out assays for the identification ofantagonists for said receptor.

[0041] Processes capable of being modulated by retinoid receptors, inaccordance with the present invention, also include the in vivomodulation of lipid metabolism, in vivo modulation of skin-relatedprocesses (e.g., acne, aging, wrinkling, skin cancer, and the like), invivo modulation of malignant cell development, such as occurs, forexample, in acute promyelocytic leukemia, testicular cancer, lungcancer, and the like. The ability of compounds of the invention tomodulate such processes is evidenced in a number of ways. See, forexample, FIG. 6 where the ability of RXR-alpha, in the presence ofligand therefor (e.g., 9-cis-retinoic acid) is shown to exert a strongeffect on the expression of genes under the control of regulatoryelements of. apolipoprotein AI. Similarly, studies with model systemsfor a variety of disease states (e.g., differentiation of HL60 cells asa model for acute promyelocytic leukemia, proliferation of melanoma celllines as a model for skin cancer, differentiation of keratinocytes as amodel for non-malignant skin disorders, and the like), as set forth inthe Examples, demonstrate the ability of retinoid receptors, in thepresence of ligand therefor, e.g., 9-cis-retinoic acid, to exert astrong effect on such disease states. Such in vivo applications of theinvention process may allow the modulation of various biologicalprocesses with reduced occurrence of undesirable side effects, and thelike.

[0042] In vivo applications of the invention process(es) (andcompositions) can be employed with a wide range of subjects, such as,for example, humans, rodents, sheep, pigs, cows, and the like.

[0043] As employed herein, the term “alkyl”, refers to “lower alkyl”,i.e., alkyl moieties having in the range of 1 up to about 4 carbonatoms, i.e., methyl groups, ethyl groups, propyl groups, isopropylgroups, normal-butyl groups, isobutyl groups, sec-butyl groups,tert-butyl groups, and the like.

[0044] Cyclic moieties contemplated as part of the compounds employed inthe practice of the present invention include 5-, 6-, and 7-memberedcarbocyclic, heterocyclic aromatic or heteroaromatic rings. Included inthis definition, for example, are optionally substituted saturated,mono-unsaturated or polyunsaturated carbocyclic species, such as, forexample, cyclopentane, cyclopentene, cyclohexane, cyclohex-2-ene,cyclohex-3-ene, cyclohex-4-ene, and cyclohex-5-ene isomers, and 2,4-,2,5-, and 3,5-cyclohexadiene variants thereof. Examples of heterocyclicspecies contemplated as part of the compounds employed in the practiceof the present invention include dihydrofuran, tetrahydrofuran,dihydrothiophene, tetrahydrothiophene, dihydropyran, tetrahydropyran,dihydrothiopyran, tetrahydrothiopyran, piperidine, pyrrolidine, and thelike, as well as derivatives thereof. Examples of aromatic orheteroaromatic species contemplated as part of the rexoid compounds ofthe present invention include phenyl, tolyl, xylyl, mesityl, benzyl,pyridyl, thiophenyl, furanyl, and the like, as well as derivativesthereof.

[0045] Preferred cyclic moieties are typically geminally di-substituted,mono-unsaturated species. Presently preferred geminally di-substituted,mono-unsaturated cyclic moieties are the 1,1,5-trisubstitutedcyclohex-5-ene structure of naturally occurring retinoic acid (i.e., thering structure of β-ionone; the position of the substituents on the ringare designated employing the traditional retinoic acid numberingconvention for the ring structure of β-ionone), as well as the1,1,4,5-tri-substituted cyclohex-5-ene structure provided by hydroxy- orketo-substituted derivatives of the traditional β-ionone structure.

[0046] Compounds contemplated for use in the practice of the presentinvention include compounds having the structure:

[0047] wherein:

[0048] unsaturation between carbon atoms C⁹ and C¹⁰ has a cisconfiguration, and one or both sites of unsaturation between carbonatoms C¹¹ through C¹⁴ optionally have a cis configuration;

[0049] “Ring” is a cyclic moiety;

[0050] Z is selected from carboxyl, carboxaldehyde, hydroxyalkyl,thioalkyl, hydroxyalkyl phosphate, alkyl ether of a hydroxyalkyl group,alkyl thioether of a thioalkyl group, esters of hydroxyalkyl groups,thioesters of hydroxyalkyl group, esters of thioalkyl groups, thioestersof thioalkyl groups, aminoalkyl, N-acyl aminoalkyl, carbamate, and thelike; and

[0051] R on each of C⁷,C⁸, C⁹, C¹⁰, ¹¹, C¹², C¹³, or C¹⁴ isindependently selected from H, halogen, alkyl, aryl, hydroxy, thiol,alkoxy, thioalkoxy, amino, or any of the Z substituents; or

[0052] any two or more of the R groups can be linked to one another toform one or more ring structures.

[0053] Presently preferred compounds which are contemplated by the abovegeneric structure include 9-cis-retinoic acid, as well as novelderivatives thereof such as-9-phenyl-9-cis-retinoic acid,4-hydroxy-9-cis-retinoic acid, 4-keto-9-cis-retinoic acid, and the like.

[0054] In another preferred embodiment of the present invention, thesubstituents on C⁹ and C¹³ are methyl; in yet another preferredembodiment, the substituents on two or more of the side chain carbons(i.e., C⁷, C⁸, C⁹, C¹⁰, C¹¹, C¹², C¹³, or C¹⁴) can be linked together toform a ring structure. For example, the substituents on C⁸ and C¹¹ canbe linked together to form a structure having a constrained 9-cis doublebond (i.e., a 9-cis locked rexoid derivative), as follows:

[0055] wherein:

[0056] X is —[(CR₂)_(x)—X′(CR₂)_(y)]—,

[0057] X′is selected from —O—, carbonyl (>CO), —S—, —S(O)—, —S(O)₂—,thiocarbonyl (>CS), —NR″—, or —CR₂—,

[0058] R, Ring and Z are as defined above,

[0059] R″ is hydrogen, alkyl, hydroxy, thiol, or alkoxy acyl(—CO—O-alkyl);

[0060] x is 0, 1 or 2,

[0061] y is 0, 1, or 2, and

[0062] x+y≦2.

[0063] Such compounds include cyclopentene derivatives, cyclohexenederivatives, cycloheptene derivatives, dihydrofuran derivatives,dihydropyrrole derivatives, and the like, wherein the cyclic structurelinking C⁸ and C¹¹ serves to prevent isomerization of the cis doublebond between C⁹ and C¹⁰.

[0064] Especially preferred derivatives of structure I are those where Zis a carboxyl group, and Ring is a β-ionone-like species having thestructure:

[0065] wherein:

[0066] each R is independently defined as provided above;

[0067] any one of C², C³, or C⁴ can be replaced with —O—, carbonyl(>CO), —S—, —S(O)—, —S(O)₂—, thiocarbonyl (>CS), or —NR″—; wherein R″ isas defined above; and

[0068] said cyclic moiety exists as the saturated, 2-ene, 3-ene, 4-ene,or 5-ene mono-unsaturated isomer; the 2,4-, 2,5-, or 3,5-dienederivative thereof; or an aromatic derivative thereof.

[0069] Especially preferred species for use in the practice of thepresent invention are derivatives of structure I where Z is a carboxylgroup, and Ring is a 1,1,5-trisubstituted cyclohex-5-ene structure or a1,1,4,5-tetrasubstituted cyclohex-5-ene structure.

[0070] Similarly, the substituents on C¹⁰ and C¹³ can be linked togetherto form a structure having a constrained 9,11-di-cis configuration(i.e., a 9-cis locked rexoid derivative), as follows:

[0071] wherein:

[0072] X, X′, R, R″, Z, Ring, x and y are as defined above.

[0073] Such compounds include cyclopentene derivatives, cyclohexenederivatives, cycloheptene derivatives, dihydrofuran derivatives,dihydropyrrole derivatives, and the like, wherein the cyclic structurelinking C¹⁰ and C¹³ serves to hinder isomerization of the cis doublebond between C⁹ and C¹⁰, and prevent isomerization of the cis doublebond between C¹¹ and C¹².

[0074] Especially preferred derivatives of Structure II are those whereZ is a carboxyl group, and the Ring is a 1,1,5-trisubstitutedcyclohex-5-ene structure or a 1,1,4,5-tetrasubstituted cyclohex-5-enestructure.

[0075] Similarly, at least two of the substituents on C⁸, C¹¹, and/orC¹⁴ can be linked together to form a structure having a constrained9,13-di-cis configuration (i.e., a 9-cis locked rexoid derivative),shown below as Structure

[0076] wherein:

[0077] one A is X and the other A is X′, and

[0078] X, X′, R, R″, Z, Ring, x and y are as defined above. Those. ofskill in the art recognize that the junction between the two bridginggroups (A) can only occur through an atom with a valence of three orfour (i.e., through carbon or nitrogen), so as to accomodate the bondsrequired to link the fused rings together.

[0079] Similarly, at least two of the substituents on C⁸, C¹¹, and/orC¹⁴ can be linked together, and further linked to C⁵ of Ring, or to asubstituent on C⁵ to form a structure having a constrained 9,13-di-cisconfiguration (i.e., a 9-cis locked rexoid derivative), shown below asStructure IV:

[0080] wherein:

[0081] one A is X and the other A is X′,

[0082] B is X′, and

[0083] X, X′, R, R″, Z, Ring, x and y are as defined above. As notedabove with respect to Structure III, those of skill in the art recognizethat the junction(s) between the bridging groups (A) and (B) can onlyoccur through an atom with a valence of three or four (i.e., throughcarbon or nitrogen), so as to accomodate the bonds required to link thefused rings together.

[0084] Such compounds include cyclopentene derivatives, cyclohexenederivatives, cycloheptene derivatives, dihydrofuran derivatives,dihydropyrrole derivatives, and the like, wherein. the cyclic structureslinking C⁸, C¹¹ and/or C¹³ serves to prevent isomerization of the cisdouble bonds at carbon 9 and carbon 13.

[0085] Especially preferred derivatives of Structures III and IV arethose where Z is a carboxyl group, and Ring is a 1,1,5-trisubstitutedcyclohex-5-ene structure or a 1,1,4,5-tetrasubstituted cyclohex-5-enestructure.

[0086] Similarly, the substituents on C¹⁰ and C¹¹ can be linked togetherto form a structure having a constrained 9-cis double bond (i.e., a9-cis locked rexoid derivative), as follows:

[0087] wherein:

[0088] X″ is —[(CR₂)_(a)—X′—(CR₂)_(b)]—,

[0089] X′, R, R″, Ring and Z are as defined above,

[0090] a is 0, 1, 2, 3 or 4,

[0091] b is 0, 1, 2, 3, or 4, and

[0092] a+b is ≧2, but ≦4.

[0093] Such compounds include cyclopentene derivatives, cyclohexenederivatives, cycloheptene derivatives, dihydrofuran derivatives,dihydropyrrole derivatives, and the like, wherein the cyclic structurelinking C¹⁰ and C¹¹ serves to prevent isomerization of the cis doublebond between C⁹ and C¹⁰.

[0094] Especially preferred derivatives of Structure V are those where Zis a carboxyl group, and Ring is a 1,1,5-trisubstituted cyclohex-5-enestructure or a 1,1,4,5-tetrasubstituted cyclohex-5-ene structure.

[0095] Similarly, the substituents on C⁷ and C⁹ can be linked together,and the substituents on C¹⁰ and C¹² can be linked together to form astructure having a constrained 9-cis double bond (i.e., a 9-cis lockedrexoid derivative), as follows:

[0096] wherein:

[0097] Y is —[(CR₂)_(c)—X′—(CR₂)_(d)]—,

[0098] X′, R, R″, Ring and Z are as defined above,

[0099] c is 0, 1, 2 or 3,

[0100] d is 0, 1, 2 or 3, and

[0101] c+d≧1 but ≦3.

[0102] Such compounds include cyclopentene derivatives, cyclohexenederivatives, cycloheptene, derivatives, dihydrofuran derivatives,dihydropyrrole derivatives, and the like, wherein the cyclic structureslinking C⁷and C⁹ and C¹⁰ and C¹² serve to prevent isomerization of thecis double bond between C⁹ and C¹⁰.

[0103] Especially preferred derivatives of Structure VI are those whereZ is a carboxyl group, and Ring is a 1,1,5-trisubstituted cyclohex-5-enestructure or a 1,1,4,5-tetrasubstituted cyclohex-5-ene structure.

[0104] Similarly, the substituents on C⁹ and C¹⁰ can be linked togetherto form a structure having a constrained C-9 double bond (i.e., a 9-cislocked rexoid derivative), as follows:

[0105] wherein:

[0106] X″′ is X″ or an unsaturated linking group having the structure:

—[Q═CR—J]—,

[0107] wherein Q is —N═ or —CR═, and J is —CR═CR—, —N═CR—, —CR═N—, —O—,—S—, or —NR″—,

[0108] thereby incorporating C⁹ and C¹⁰ of the rexoid compound into anaromatic (or pseudo-aromatic) ring, and

[0109] X′, X″, R, R″, Ring, Z, a and b are as defined above.

[0110] Such compounds include cyclohexene derivatives, cycloheptenederivatives, benzene derivatives, pyridine derivatives, furanderivatives, thiophene derivatives, pyrrole derivatives, oxazole,derivatives, thiazole derivatives, imidazole derivatives, pyrazolederivatives, and the like, wherein the cyclic structure linking C⁹ andC¹⁰ serves to prevent isomerization of the C⁹ -C¹⁰ double bond; however,rotation about the 8-9 and/or 10-11 single bonds can still occur.

[0111] Especially preferred derivatives of Structure VII are those whereZ is a carboxyl group, and Ring is a 1,1,5-trisubstituted cyclohex-5-enestructure or a 1,1,4,5-tetrasubstituted cyclohex-5-ene structure.

[0112] In addition to the structures set forth above, those of skill inthe art can readily identify additional means to constrain the basiccis-configuration containing rexoid compounds employed in the practiceof the present invention.

[0113] In accordance with a preferred embodiment of the presentinvention, the cyclic moiety has the β-ionone structure set forth above.Especially preferred are the 1,1,5-trisubstituted cyclohex-5-enestructure (characteristic of β-ionone) as well as the closely related1,1,4,5-tetrasubstituted cyclohex-5-ene structure from which many rexoidcompounds according to the present invention can be prepared.

[0114] In accordance with a particularly preferred embodiment of thepresent invention, the compounds employed in the invention process areselected from 9-cis-retinoic acid and derivatives thereof ascontemplated by Structure A set forth above, as well as 9-cis-lockedderivatives of retinoic acid as set forth in Structures I-VII above.Examples of specific compounds contemplated for use in the practice ofthe present invention are compounds wherein Z is carboxy, Ring is the1,1,5-trisubstituted cyclohex-5-ene structure characteristic of β-ionone(or the closely related 1,1,4,5-tetrasubstituted cyclohex-5-ene), andhaving a side chain structure(s) as described above for StructuresI-VII.

[0115] “Rexoid” derivatives as described above can be prepared employinga variety of synthetic methods, which are readily available (and wellknown) to those of skill in the art. See, for example, the methodsdescribed in Chemistry and Biology of Synthetic Retinoids, Dawson andOkamura, eds., CRC Press, Inc. (1990), especially Chapter 4, by Ito(found at pages 78-97), and Chapter 9, by de Lera et al. (found at pages202-227) can readily be adapted for the preparation of the compoundsdescribed herein. The contents of this publication are herebyincorporated by reference herein. See also Asato et al., J. Am. Chem.Soc. 108: 5032 (1986); Sheves et al., J. Am. Chem. Soc. 108: 6440(1986); Akita et al., J. Am. Chem. Soc. 102: 6370 (1980); Derguini andNakanishi, Photobiochem. and Photobiophys. 13: 259 (1986), the entirecontents of each of which is hereby incorporated by reference herein.

[0116] In accordance with another embodiment of the present invention,there is provided a method for modulating processes mediated by retinoidreceptors, said method comprising conducting said process in thepresence of:

[0117] (a) at least one compound of the structure:

[0118] wherein:

[0119] each site of unsaturation in the side chain comprising carbonatoms C⁷ through C¹⁴ has a trans configuration;

[0120] “Ring”, Z, and R are as previously described, and

[0121] (b) a cis/trans isomerase capable of converting at least the9-double bond from the trans configuration to the cis-configuration.

[0122] As employed herein, the term “cis/trans isomerase” refers toenzymes which promote a change of geometrical configuration at a doublebond. Examples of such enzymes include maleate isomerase,maleylacetoacetate isomerase, retinal isomerase, maleylpyruvateisomerase, linoleate isomerase, furylfuramide isomerase, and the like.

[0123] In accordance with yet another embodiment of the presentinvention, there is provided a method to produce compound(s) of thestructure:

[0124] wherein:

[0125] unsaturation between carbon atoms C⁹ and C¹⁰ has a cisconfiguration, and one or both sites of unsaturation between carbonatoms C¹¹ through C¹⁴ optionally have a cis configuration;

[0126] “Ring” is a cyclic moiety;

[0127] Z is selected from carboxyl, carboxaldehyde, hydroxyalkyl,thioalkyl, hydroxyalkyl phosphate, alkyl ether of a hydroxyalkyl group,alkyl thioether of a thioalkyl group, esters of hydroxyalkyl groups,thioesters of hydroxyalkyl group, esters of thioalkyl groups, thioestersof thioalkyl groups, aminoalkyl, N-acyl aminoalkyl, carbamate, and thelike; and

[0128] each R is independently selected from H, halogen, alkyl, aryl,hydroxy, thiol, alkoxy, thioalkoxy, amino, or any of the Z substituents;

[0129] from the corresponding all-trans configuration material, saidmethod comprising contacting said all-trans configuration material witha cis/trans isomerase under isomerization conditions.

[0130] In accordance with still another embodiment of the presentinvention, there are provided novel compositions comprising compound(s)of Structure A (excluding previously identified compounds such asretinoic acid as well as constrained. compounds selected from StructuresI-VII, as set forth above. Examples of such compounds include9-phenyl-9-cis-retinoic acid, 4-hydroxy-9-cis-retinoic acid,4-keto-9-cis-retinoic acid, and the like. Presently preferred compoundsare those wherein Z is carboxyl and Ring is a 1,1,5-trisubstitutedcyclohex-5-ene structure or a 1,1,4,5-tetrasubstituted cyclohex-5-enestructure.

[0131] The invention compounds can be employed for both in vitro and invivo applications. For in vivo applications, the invention compounds canbe incorporated into a pharmaceutically acceptable formulation foradministration. Those of skill in the art can readily determine suitabledosage levels when the invention compounds are so used.

[0132] As employed herein, the phrase “suitable dosage levels” refers tolevels of compound sufficient to provide circulating concentrations highenough to effect activation of retinoid receptor(s). Such aconcentration typically falls in the range of about 10 nM up to 2 μM;with concentrations in the range of about 100 nM up to 200 nM beingpreferred.

[0133] In accordance with a particular embodiment of the presentinvention, compositions comprising at least one 9-cis-retinoic acid-likecompound (as described above), and a pharmaceutically acceptable carrierare contemplated. Exemplary pharmaceutically acceptable carriers includecarriers suitable for oral, intravenous, subcutaneous, intramuscular,intracutaneous, and the like administration. Administration in the formof creams, lotions, tablets, dispersible powders, granules, syrups,elixirs, sterile aqueous or non-aqueous solutions, suspensions oremulsions, and the like, is contemplated.

[0134] For the preparation of oral liquids, suitable carriers includeemulsions, solutions, suspensions, syrups, and the like, optionallycontaining additives such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring and perfuming agents, and the like.

[0135] For the preparation of fluids for parenteral administration,suitable carriers include sterile aqueous or non-aqueous solutions,suspensions, or emulsions. Examples of non-aqueous solvents or vehiclesare propylene glycol, polyethylene glycol, vegetable oils, such as oliveoil and corn oil, gelatin, and injectable organic esters such as ethyloleate. Such dosage forms may also contain adjuvants such as preserving,wetting, emulsifying, and dispersing agents. They may be sterilized, forexample, by filtration through a bacteria-retaining filter, byincorporating sterilizing agents into the compositions, by irradiatingthe compositions, or by heating the compositions. They can also bemanufactured in the form of sterile water, or some other sterileinjectable medium immediately before use.

[0136] The invention will now be described in greater detail byreference to the following non-limiting examples.

EXAMPLES Example 1 Identification of Compound(s) that Activate RXR

[0137] In order to a certain if retinoic acid can be converted to aproduct that binds directly to RXR, thereby resulting in modulation oftranscription, a strategy was developed to identify retinoic acidmetabolites that might modulate the transcriptional properties of RXR.The identification of any such active metabolite would allow one tofurther determine whether this metabolite was capable of directlybinding to the receptor protein.

[0138] Accordingly, the Drosophila melanogaster Schneider cell line (S2)was incubated with or without all-trans-retinoic acid (RA) for a periodof 24 hours. Prior to the addition of retinoic acid, Drosophilamelanogaster Schneider cell line (S2) cells were grown in SchneiderDrosophila medium (GIBCO) supplemented with penicillin, streptomycin and12% heat inactivated FCS (Irvine Scientific). One hundred tissue cultureflasks (75 cm²) were set up with 10⁷ cells and 12 ml of medium/flask.Twenty four hours later, either all-trans-retinoic acid (or ethanolsolvent control) was added to each flask to a final concentration of5×10⁻⁶ M in reduced light conditions. Cells were harvested 24 hourslater by centrifugation for 5 minutes at 800 g. Cells were washed twicewith PBS and the resultant pellets were frozen at −80° C. untilextraction.

[0139] In parallel, CV-1 cells were set up on 64 tissue culture dishes(150 mm) at 2×10⁶ cells and 25 ml of medium/dish. Cells were treatedwith retinoic acid and harvested as with the S2 cells except that theCV-1 cells (which are adherent) were washed in their dishes with PBS andscraped with a rubber policeman prior to centrifugation and freezing.

[0140] Following incubation, the cell pellets were collected,organically extracted and chromatographically fractionated by HPLC. Thevarious HPLC fractions were assayed for their ability to produce aligand dependent increase in transcriptional activity mediated by RXR.This assay system involves transfecting cells with the cDNA for the RXRreceptor and a luciferase reporter molecule which is under control of apromoter containing a RXR response element (RXRE) [see Mangelsdorf etal., Cell 66:555 (1991)]. The addition of a ligand capable of activatingRXR results in an increase in luciferase activity.

[0141] Schneider cells, CV-1 cells and mouse tissues were extracted asdescribed by C. Thaller and G. Eichele in Nature Vol. 327:625 (1987).Mouse tissue was used to determine if any RXR ligand is present in vivo.In the case of tissue extractions, 2.10⁵ dpm internal standard[11,12-³H]-all-trans-retinoic acid (New England Nuclear) or9-cis-retinoic acid (generated by isomerization with light) were addedto the homogenate. Extracts were fractionated on a Waters Novapak 300 mmC₁₈ analytical column at a flow rate of 1 ml min⁻¹. The mobile phase (G)was a 1:1 mixture of:

[0142] A [CH₃CN/CH₃OH/2% aqueous CH₃COOH (3:1:1)] and

[0143] E [CH₃CN/CH₃OH/2% aqueous CH₃COOH (11:3:10)].

[0144] Other mobile phases used have the following compositions:

[0145] C: CH₃CN/CH₃OH/H₂O/CH₃COOH (80:10:10:1),

[0146] H: mix CH₃OH/10 mM ammonium acetate (9:1) with equal volume ofCH₃OH/10 mM ammonium acetate (3:1).

[0147] Methyl esters of retinoic acid isomers and/or metabolitescontained in the HPLC fractions were generated as described in Wedden etal. [(Meth. Enzymol. 190:201 (1990)]. Reference standards used were fromAldrich, Sigma or kindly provided by Hoffmann-LaRoche. Authentic9-cis-retinol, 9-cis-retinoic acid and 9-cis-methylretinoate were eithersynthesized from 9-cis-retinal (see E. J. Corey et al., J. Am. Chem.Soc. 90:5616 (1968); C. D. B. Bridges & R. A. Alvares [(Meth. Enzymol.81:463 (1982)] or generated by photoisomerization of the all-transisomer followed by fractionation of the resulting isomers by HPLC.

[0148] Photoisomerization of all-trans-retinoic acid is carried outemploying standard isomerization techniques which are well known tothose of skill in the art. For example, retinoic acid can be dissolvedin a polar organic solvent such as ethanol, placed in a quartz cuvette,and irradiated with a variety of wavelengths of light (such asfluorescent light). Temperature at which irradiation is carried out isnot critical; accordingly, irradiation can be carried out at roomtemperature. Irradiation time is also not critical; typical irradiationtimes are in the range of about 0.5-2 hours.

[0149] The various HPLC fractions were diluted 1:100 and assayed fortheir ability to modulate the transcriptional properties of RXR.

[0150] Cotransfection Assay in CV-1 Cells

[0151] A monkey kidney cell line, CV-1, was used in the cis-trans assay.Cells were transfected with two DNA transfection vectors. Thetrans-vector allowed efficient production of retinoid receptor (e.g.,RAR or RXR) in these cells, which do not normally express thesereceptors. The cis-vector contains an easily assayable gene, in thiscase the firefly luciferase, coupled to a retinoid-responsive promoter.Addition of retinoic acid or an appropriate synthetic retinoid resultsin the formation of a retinoid-receptor complex that activates theluciferase gene, causing light to be emitted from cell extracts. Thelevel of luciferase activity is directly proportional to theeffectiveness of the retinoid-receptor complex in activating geneexpression. This sensitive and reproducible cotransfection approachpermits the identification of retinoids that interact with the differentreceptor isoforms.

[0152] Cells were cultured in DMEM supplemented with 10% charcoalresin-stripped fetal bovine serum, and experiments were conducted in96-well plates. The plasmids were transiently transfected by the calciumphosphate method [Umesono and Evans, Cell 57:1139-1146 (1989); Berger etal., J. Steroid Biochem. Molec. Biol. 41:733-738 (1992)] by using 10 ngof a pRS (Rous sarcoma virus promoter) receptor-expression plasmidvector, 50 ng of the reporter luciferase (LUC) plasmid, 50 ng ofPRSβ-GAL (β-galactosidase) as an internal control, and 90 ng of carrierplasmid PGEM. Cells were transfected for 6 hours and then washed toremove the precipitate. The cells were then incubated for 36 hours withor without retinoid. After the transfection, all subsequent steps wereperformed on a Beckman Biomek Automated Workstation. Cell extracts wereprepared as described by Berger et al. supra, then assayed forluciferase and β-galactosidase activities. All determinations wereperformed in triplicate in two independent experiments and werenormalized for transfection efficiency by using β-galactosidase as theinternal control. Retinoid activity was normalized relative to that ofretinoic acid and is expressed as potency (EC50), which is theconcentration of retinoid required to produce 50% of the maximalobserved response, and efficacy (%), which is the maximal responseobserved relative to that of retinoic acid at 10⁻⁵ M.

[0153] The receptor expression vectors used in the cotransfection assayhave been described previously [pRShRAR-α: Giguere et al., Nature330:624-629 (1987); pRShRAR-β and pRShRAR-γ: Ishikawa et al., Mol.Endocrinol. 4:837-844 (1990); retinoid X receptor-alpha (RXR-α) [seeMangelsdorf et al., in Nature 345: 224-229 (1990)], retinoid Xreceptor-beta (RXR-β) and retinoid X receptor-gamma (RXR-γ) [seeMangelsdorf et al., Genes and Development 6:329-344 (1992)]. A basalreporter plasmid ΔMTV-LUC [Hollenberg and Evans, Cell 55:899-906 (1988)]containing two copies of the TRE-palindromic response element5′-TCAGGTCATGACCTGA-3′ [SEQ ID No 2; see Umesono et al., Nature336:262-265 (1988)] was used in all transfections for the retinoidreceptors.

[0154] The bacterial expression vector for PET-8c-RAR-α used in thecompetitive binding assay has been reported (Yang et al., Proc. Natl.Acad. Sci. USA 88:3559-3563 (1991)). Similar expression vectorsemploying the PET-8c vector system [Studier et al., Methods inEnzymology 185:60-69 (1990)] were constructed for RAR-β and RAR-γ.

[0155] The transactivation profile of RXR-alpha with the various HPLCfractions containing various retinoic acid isomers and/or metabolites isshown in FIG. 1. These data reveal two distinct regions. of activity,one relatively early (fraction 7) and a second broader region ofactivity (fractions 16-21) that elutes considerably later. Theall-trans-retinoic acid coelutes in fractions 20 and 21 (FIG. 1) and isthe major U.V. absorbing material present in the cell extracts. However,the activity profile demonstrates that, in addition toall-trans-retinoic acid, there are active components that must bederived from, or induced by, all-trans-retinoic acid that activate RXR.

[0156] To identify potential compounds that would be as effective ormore active than all-trans-retinoic acid, one must take into account notonly the activity of the individual fractions, but also theirconcentrations. All active fractions were therefore reassayed over abroad range of concentrations, taking into account the relativeconcentrations of the individual fractions. To determine the relativeconcentrations of the fractions, the following initial assumptions weremade: 1) the active fractions are retinoic acid metabolites and 2) themolar extinction coefficient of the various active fractions isrelatively similar (i.e., within a factor of two). This assumption issupported by values reported in the literature for a large number ofretinoids. A comparison of the transactivation profile ofall-trans-retinoic acid, (i.e., fraction 20) on RAR-alpha and RXR-alphais shown in FIG. 2a. Although the maximal activation (i.e., efficacy) ofRAR and RXR with retinoic acid is similar, RAR is more sensitive by afactor of approximately 10 fold (i.e., 10 fold more potent). Incontrast, analysis of the various fractions produced as describes abovedemonstrates that fraction 18 is considerably more active on RXR thanRAR (see FIG. 2b). These data suggest that a metabolic product presentin S2 cells pretreated with retinoic acid is a more potent activator ofthe RXR subfamily than the RAR subfamily.

Example 2 Identification of 9-cis Retinoic Acid as a Transactivator ofRXR

[0157] Two observations suggest that fraction 18 (peak X, see FIG. 1) isa cellular metabolite of all-trans-retinoic acid. First, extracts ofSchneider cells grown in the absence of all-trans-retinoic acid do notexhibit peak X. Second, when cells are exposed to all-trans-retinoicacid, X appears in a time-dependent fashion.

[0158] Therefore, to chemically identify X, fraction 18 was subjected tochemical derivatization, high performance liquid chromatography (HPLC)and gas chromatography/mass spectrometry (GC/MS). It was found that uponmethylation with diazomethane, the retention time of peak X shiftsdramatically (i.e., from 10.2 minutes to 19.5 minutes under the HPLCconditions used). This indicates that the compound(s) corresponding topeak X has a free carboxyl group. When methylated X was analyzed byGC/MS, the electron impact mode revealed that X gives rise to amolecular ion at m/z 314, corresponding to that of a retinoic acidmethyl ester. This suggests that X is a stereoisomer of retinoic acid.To determine which isomer X represents, the retention time of X wascompared with that of 9-cis-, 11-cis- and 13-cis-retinoic acid. It wasfound that X coelutes with authentic 9-cis-retinoic acid. Furthermore,the methyl ester of X coelutes with 9-cis-methylretinoate, and when themethyl ester of X is reduced to the alcohol with lithium aluminumhydride, the resulting product coelutes with authentic 9-cis-retinol.

[0159] For GC/MS analysis, methylated retinoic acid isomers weredissolved in hexane. The sample was injected via a falling needleinjector (280° C.) into a 30 m×0.32 mm fused silica DB5 capillary column(J+J scientific) inserted directly into the ion source of a VG Trio-1000mass spectrometer operating in electron impact mode (70 eV). The samplewas eluted with a temperature gradient (200-300° C., 10° C. min⁻¹).

[0160] Finally, the mass spectrum of authentic 9-cis-retinoic acidmethyl ester and that of methylated peak X are found to be identical.Taken together these analyses establish that peak X represents9-cis-retinoic acid. Although earlier work indicated the presence of9-cis-retinol in fish liver, it was not clear whether 9-cis-retinoicacid existed in vivo (i.e., whether 9-cis-retinoic acid is aphysiological compound). To find out if 9-cis-retinoic acid exists invivo, mouse liver and kidney tissues were extracted. These tissues wereselected because they contain a. broad spectrum of retinoid metabolitesand also express RXR. Prior to extraction, radiolabeled 9-cis-retinoicacid was added to the kidney homogenate to serve as an internalstandard. Extracts were first fractionated on a reverse phase column(Waters Novo pak 300 mm C₁₈ analytical column at a flow rate of 1ml/min) using mobile phase G.

[0161] Fractions from the kidney extracts containing radioactiveinternal standard were rechromatographed on a second C₁₈ column usingmobile phase H. This procedure gave a small, but distinct absorbancepeak which co-migrated with authentic 9-cis-retinoic acid.

[0162] Similarly, liver extract was fractionated on a reverse .phasecolumn and eluted with mobile phase G. However under the conditionsemployed, 9-cis-retinoic acid eluted with all-trans-retinol (which isabundantly present in the liver). To separate these two retinoids, thisfraction was methylated with diazomethane and then reanalyzed by HPLCemploying mobile phase C. This approach resulted in a distinct peakcoeluting with the authentic methyl ester of 9-cis-retinoic acid.

[0163] To rule out the possibility that 9-cis-retinoic acid had formedduring the extraction procedure from all-trans-retinoic acid, livertissue homogenate was spiked with tritiated all-trans-retinoic acid.Subsequent HPLC fractionation revealed that 94% of the radioactivitystill resided in all-trans-retinoic acid, approximately 5% in13-cis-retinoic acid and 1% or less in 9-cis-retinoic acid. Based onpeak area integration the concentrations of 9-cis-retinoic acid in liverand kidney are estimated to be ˜4 ng, and ˜4 ng, respectively, per g ofwet weight. This indicates that endogenous 9-cis-retinoic acid is notformed from all-trans-retinoic acid during extraction. In conclusion,these experiments establish that 9-cis-retinoic acid is a naturallyoccurring retinoic acid isomer.

Example 3 Transactivation Profile of Retinoid Isomers on RXR and RAR

[0164] The establishment that peak X represents a stereoisomer ofall-trans-retinoic acid suggested that the various retinoid isomers mayhave different retinoid receptor activation profiles. To further analyzethe ability of retinoic acid isomers to modulate the transcriptionalproperties of RXR-alpha and RAR-alpha, the four major photoisomers ofall-trans-retinoic acid were identified and assayed for the ability totransactivate RXR and RAR. FIG. 3 shows the dose response curves for13-cis-, 11-cis-, 9-cis- and all-trans-retinoic acid for both RAR-alphaand RXR-alpha.

[0165] Of the four major isomers of retinoic acid, 9-cis-retinoic acidis seen to be the most potent and efficacious activator of RXR-alpha inboth insect S2 cells (see FIG. 3A) and mammalian CV-1 cells (see FIG.3B). The maximal response (EC50 value) is 10⁻⁸ M and 5×10⁻⁸ M,respectively. The observed rank order of potency for the differentisomers is the same in both cell lines. 9-cis-retinoic acid isapproximately 40 fold more potent as an activator of RXR than 11-cis-,13-cis- or all-trans-retinoic acid. These transactivation data stronglysuggest that 9-cis-retinoic acid is an endogenous RXR-alpha activator.

[0166] In contrast, 9-cis-retinoic acid is equipotent toall-trans-retinoic acid as an activator of RAR-alpha (FIG. 3C). The EC50value for 9-cis-retinoic acid on RAR-alpha is 2×10⁻⁷ M. 9-cis-retinoicacid is the most potent RXR-alpha ligand to be tested to date.

[0167] Similarly, transactivation of other isoforms of RXR (i.e.,RXR-beta, RXR-gamma) and RAR (i.e., RAR-beta, RAR-gamma) by9-cis-retinoic acid was also examined. 9-cis-retinoic acid was alsofound to be a potent activator of these isoforms as well, as shown inTable 1: TABLE 1 EC₅₀* (nM) Receptor All-trans-retinoic Acid9-cis-retinoic Acid PAR-α 3861 ± 13 327 ± 30 RAR-β  152 ± 12  95 ± 13PAR-γ  48 ± 8   61 ± 5  RXR-α 1174 ± 26 255 ± 17 RXR-β 1841 ± 26 218 ±17 RXR-γ 1369 ± 26 254 ± 19

Example 4 9-cis Retinoic Acid Binds Directly to RXRs

[0168] The ability of 9-cis-retinoic acid to transactivate RXR-alphasuggested testing to see whether 9-cis-retinoic acid was also capable ofbinding directly to RXRs. RXR-alpha was expressed in baculovirus and wasshown to have biochemical properties that were identical to themammalian expressed protein. The baculovirus expressed protein had amolecular weight of 51,000, reacted specifically with RXR-alpha antibodyand was capable of binding in vitro to DNA sequences that have beenpreviously shown to be specific RXR response elements (i.e. CRBPII, seeMangelsdorf et al., Cell 66:555 (1991); apolipoprotein AI gene, seeRottman et al., Mol. Cell Biol. 11:3814 (1991)].

[0169] To characterize the ligand binding characteristics of9-cis-retinoic acid to baculovirus-derived RXR, saturation bindinganalysis was carried out (see FIG. 4). Radiolabelled 9-cis-retinoic acidbinds specifically to RXR-alpha in a saturable manner. Scatchardanalysis suggests a single high affinity binding site with a Kd value of11.7 nM (see FIG. 4b). Under identical binding conditions[³H]-all-trans-retinoic acid did not bind to RXR-alpha (see FIG. 4a). Inaddition, 9-cis-retinoic acid was also capable of binding specificallyto RAR-alpha as a high affinity ligand. 9-cis-retinoic acid did not bindto mock baculovirus extracts (i.e., control extracts from cells that donot express RXRs).

[0170] Similarly, binding studies were also carried out with otherisoforms of RXR (i.e., RXR-beta, RXR-gamma), other isoforms of RAR(i.e., RAR-beta, RAR-gamma), and cellular retinoic acid binding protein(CRABP) with all-trans-retinoic acid and 9-cis-retinoic acid. Whileall-trans-retinoic acid is known to bind to each of these “receptors”,9-cis-retinoic acid was also found to bind to the other isoforms ofretinoid receptors (but not to the cellular retinoic acid bindingprotein, CRABP), as shown in Table 2: TABLE 2 Kd (nM) ReceptorAll-trans-retinoic Acid 9-cis-retinoic Acid PAR-α 0.4 0.3 PAR-β 0.4 0.2RAR-γ 0.2 0.8 RXR-α No binding 1.5 RXR-β No binding 2.1 RXR-γ No binding1.9 CRABP 20 >100

[0171] The properties of many members of the steroid hormone receptorsuperfamily have been characterized and defined using DNA cellulosechromatography [see, for example, Pike and Haussler, Proc. Natl. Acad.Sci. USA 76:5485 (1979) and Pike et al., J. Biol. Chem. 258:1289(1983)]. Receptors, such as the VDR, have been shown in the presence oftheir cognate ligand to bind to DNA-cellulose [see, for example,Allegretto et al., J. Biol. Chem. 262:1312 (1987)] with high affinityand the ligand-receptor complex elutes with a salt gradient. ADNA-cellulose column profile of the baculovirus expressed RXR that hadbeen prelabeled with [³H]-9-cis-retinoic acid is shown in FIG. 5. Thetwo different profiles represent 1) the total amount of[³H]-9-cis-retinoic acid bound and 2) the level of binding that remainsin the presence of 200-fold excess of cold (i.e. non-labeled9-cis-retinoic acid).

[0172] There is a peak of radioactivity (marked in the Figure by anarrow) that elutes off the DNA-cellulose column at 0.15 M KCl. Thiselution profile is similar to that seen with RARα in the presence of[³H]-all-trans-retinoic acid. A 200 fold excess of cold ligand (i.e.non-specific) is capable of competing greater than 90% of the totalradioactivity bound, demonstrating that the radioactivity in the peakfractions is 9-cis-retinoic acid specifically bound to RXR.

[0173] The radioactivity eluted off the column was extracted withorganic solvent and subjected to HPLC analysis.

[0174] Inspection of FIG. 5b makes it clear that the radioactivity boundto RXR co-chromatographs with authentic 9-cis-retinoic acid. Thisobservation further confirms that [³H]-9-cis-retinoic acid is thespecies bound to RXR.

[0175] To demonstrate that the protein contained in the peak fractionsis indeed RXR, these fractions (labelled 1-15 in FIG. 5a) were subjectedto immunoblot analysis using an RXRα specific polyclonal antiserum (seeFIG. 5a, top). All fractions containing radioactivity display a distinctRXRα band at a M_(r) of 51,000. When a similar experiment was conductedwith a baculovirus mock extract, no specific radioactivity was retainedon the column. Taken together, these data strongly. suggest that9-cis-retinoic acid is capable of binding specifically to RXR.

[0176] Protein samples were resuspended in 2× sample buffer [Laemelli,Nature Vol. 227:680(1970)] and boiled for 5 minutes prior to loadingonto a 9% SDS polyacrylamide gel. After electrophoretic separation thegels were electroblotted onto nitrocellulose membranes (Scheicher andSchuell) for 8 hours at 30 volts using a Hoeffer electro-transferapparatus. Membranes were then incubated in 10% isopropanol, 10% aceticacid for 15 minutes, washed 5 minutes in deionized H₂O and 5 minutes inT-TBS buffer (10 mM Tris pH 7.5, 150 mM NaCl and 0.5% Triton X-100). Themembranes were blocked in 5% nonfat milk in T-TBS for 1 hour. Theremainder of the protocol was adapted from the Amersham ECL (EnhancedChemiluminescence) Western blotting detection system kit. The primaryantibody was a rabbit polyclonal serum raised against a syntheticpeptide corresponding to amino acids 214-229 of hRXRα[Kliewer et al.,Proc. Natl. Acad. Sci. USA 89:1448-1452 (1992)]. The primary antiserumwas diluted 1:5000 in T-TBS. The secondary antibody (Donkey anti rabbitIgG conjugated to horseradish peroxidase, Amersham) was used at adilution of 1:2500.

Example 5 Effects of Topical Application of 9-cis-retinoic Acid(Compared with All-trans-retinoic Acid) on Horn-filled Utriculus Size inthe Rhino Mouse

[0177] All-trans-retinoic acid is known to influence celldifferentiation and exert profound therapeutic benefits in the treatmentof keratinization disorders [Elias et al., Arch. Dermatol.. Vol.117:160-180 (1981)]. Mezick et al. [see J. Invest. Derm. Vol. 83:110-113(1984)] demonstrated that topical treatment of rhino mice (hr hr) withall-trans-retinoic acid could reduce keratinized pilosebaceousstructures (horn-filled utriculus). This animal test model was used toevaluate the “antikeratinizing” effects of 9-cis-retinoic acid. Resultsare summarized in Table 3: TABLE 3 Pilosebaceous structure size (%red′n) Vehicle Control 178 μm 9-cis-retinoic acid, 0.1%  52 μm (−74%)0.01%  72 μm (−64%) All-trans-retinoic acid, 0.1%  44 μm (−78%) 0.01% 50 μm (−75%)

[0178] 9-cis-retinoic acid reduced the mean utriculi diameter after 14days of topical application. These results demonstrate that topicalapplication of. 9-cis-retinoic acid over a 14 day period can reducekeratinized pilosebaceous structures (horn-filled utriculus) in Rhinomouse skin. Reduction in the mean utriculi diameter by 9-cis-retinoicacid was comparable to that observed with all-trans-retinoic acid.

Example 6 Effects of 9-cis-retinoic Acid (Compared withAll-trans-retinoic Acid) on Differentiation of HL60 Cells

[0179] Retinoids are known to differentiate human promyelocytic leukemiacells. Differentiation of HL60 cells (a model system for promyelocyticleukemia) can be assessed by Nitro Blue Tetrazolium (NBT) dye reduction(superoxide anion generation) and by measurement of up-regulation of thegene encoding the β subunit of the leukocyte adherence receptor, CD18(J. B. C. vol. 263 No. 27, pp. 13863-13867).

[0180] The EC-50 for 9-cis-retinoic acid-mediated differentiation, asdetermined by NBT after 6 days treatment, was 0.2 μM compared to 2 μMfor all-trans-retinoic acid. Maximal effects (efficacies) werecomparable, and CD18 was up-regulated by both ligands. Alpha-interferonpotentiated both all-trans-retinoic acid and 9-cis-retinoicacid-mediated differentiation, as determined by NBT.

[0181] HL60R cells have been shown to be resistant to differentiation byall-trans-retinoic acid, probably related to a mutation in the retinoicacid receptor-alpha gene. This cell line was found to be resistant todifferentiation (NBT) by both all-trans-retinoic acid and 9-cis-retinoicacid at concentrations up to 10 μM.

[0182] 9-cis-retinoic acid effects differentiation of HL60 cells asevidenced by NBT and up-regulation of CD18. Compared with all-transretinoic acid, 9-cis retinoic acid is more potent with similar efficacy.

Example 7 Effects of 9-cis-retinoic Acid (Compared withAll-trans-retinoic Acid) on In Vitro Proliferation of Melanoma CellLines

[0183] All-trans-retinoic acid and several synthetic analogs (retinoids)have been shown to prevent the development of benign and malignant,chemically induced epithelial tumors in vivo [Sporn et al., Fed. Proc.Vol. 35:1332-1338 (1976)]. Lotan et al. (J. Natl. Cancer, Vol.60:1035-1041, 1978) found that all-trans-retinoic acid inhibited thegrowth of several tumor cell lines in vitro. In view of these earlierfindings, it was of interest to evaluate the growth inhibitoryproperties of 9-cis-retinoic acid.

[0184] 9-cis-retinoic acid inhibited the growth of the murine melanomacell line Clone M3 in a concentration dependent manner, as follows: %Growth inhibition (Conc added) 1 μM 0.01 μM 9-cis-retinoic acid −85%−49% all-trans-retinoic acid −94% −48%

[0185] Similarly, 9-cis retinoic acid inhibited the growth of the humanprimary metastatic melanoma cell line c81-46c in a concentrationdependent manner. % Growth inhibition (Conc added) 1 μM 0.01 μM9-cis-retinoic acid −45% −28% all-trans-retinoic acid −44% −17%

[0186] In summary, 9-cis-retinoic acid has been shown to inhibit the invitro proliferation of murine melanoma cell line Clone M3 and humanmetastatic melanoma cell line c81-46c in a concentration dependentmanner. 9-cis-retinoic acid has an equal inhibitory effect on thesecells as compared to all-trans-retinoic acid.

Example 8 Effects of 9-cis-retinoic Acid (Compared withAll-trans-retinoic Acid) on Differentiation of F9 Cells

[0187] Retinoids are known to differentiate mouse teratocarcinoma cells(F9). Differentiation of F9 cells is specifically associated withirreversible changes in morphology and induction of the biochemicalmarker alkaline phosphatase (ALP) and tissue plasminogen activator (tPA)(Biochem. J. Vol. 274:673-678).

[0188] Both all-trans-retinoic acid and 9-cis-retinoic acid induceddifferentiation of F9 cells into partial endoderm-like cells asindicated by irreversible changes in cellular morphology.All-trans-retinoic acid was 40 times more potent than 9-cis-retinoicacid in inducing ALP, maximal responses were similar.

[0189] The response of tissue plasminogen activator factor was less for9-cis-retinoic acid than for all-trans-retinoic acid. At a concentrationof 1 μM of 9-cis-retinoic acid (or all-trans-retinoic acid), increasedcellular activities of tPA by 0.48±0.05 and 0.80±0.08, respectively wereobserved. This effect was concentration-dependent.

[0190] In summary, 9-cis-retinoic acid promoted differentiation of F9cells as evidenced by changes in morphology and marker enzymeactivities. Compared with all-trans-retinoic acid, 9-cis-retinoic acidwas less potent with regard to both enzyme markers. Efficacy wascomparable with ALP but indeterminate for tPA.

Example 9 Effects of 9-cis-retinoic Acid (Compared withAll-trans-retinoic Acid) on Differentiation of Keratinocytes

[0191] Retinoids are known to inhibit squamous cell differentiation ofcultured normal human epidermal keratinocytes (NHEK534 cell line), asjudged by morphological alterations and inhibition of induction oftransglutaminase (Type I) (J. Biol. Chem. Vol. 261:15097, 1986; Lab.Invest. Vol. 56:654, 1987).

[0192] Both all-trans-retinoic acid and 9-cis-retinoic acid inhibitedsquamous cell differentiation in a concentration dependent manner asjudged by morphological changes and by transglutaminase activity. TheEC50s for inhibition of differentiation by all-trans-retinoic acid and9-cis-retinoic acid were identical (20±2.8 nM). 9-cis retinoic acid andall-trans-retinoic acid EC50s and potencies were nearly identical foreffects on transglutaminase activities.

[0193] In summary, like all-trans-retinoic acid, 9-cis-retinoic acidinhibits morphological differentiation of NHEK534 cells and induction oftransglutaminase activity.

Example 10 Synthesis of 9-Phenyl-9-cis-retinoic Acid

[0194] To a solution of 44 mg (0.10 mmole) of the following phosphonatereagent:

[0195] in THF (0.5 ml) at room temperature was added NaH (60% in oil, 5mg; 0.13 mmole) and the mixture stirred at that temperature for 10minutes. To this, 26 mg (0.08 mmole) of the aldehyde:

[0196] in THF (0.5 ml) was added at room temperature and the mixtureallowed to stir for 30 minutes. Aqueous workup in the usual manner(NH₄Cl (aq), H₂O, brine, MgSO₄) gave a mixture of 9-phenyl-9-cis esterand 9-phenyl-9,13-dicis ester (30 mg, 92%) (the calculated ratio of9-cis : 9,13-dicis=4:1).

[0197] Ethyl Ester of 9-phenyl-9-cis-retinoic Acid:

[0198] Ethyl Ester of 9-phenyl-9,13-dicis-retinoic Acid:

[0199] To a mixture of 9-cis and 9,13-dicis ester (20 mg, 0.05 mmole) inmethanol (0.7 ml) and H₂O (0.7 ml) at 25° C. was added KOH (14.3 mg,0.25 mmole). Consequently, the mixture was heated to 70° C. for 2 hours.The reaction was then cooled down to 0° C., diluted with 10 ml ofdiethyl ether), and acidified with HCl (0.12M in HCl, 2.17 ml). Aqueousworkup in the usual manner (H₂O, brine, MgSO₄) gave a mixture of 9-cisand 9,13-dicis acid. Flash column chromatography (silica, 13% ethylacetate in benzene) gave pure 9-phenyl-9-cis retinoic acid (14.5 mg.100%).

[0200] The ¹HNMR spectrum of 9-phenyl-9-cis retinoic acid is as follows:

[0201]¹HNMR (400 mHz), CDCl₃) δ 7.4-7.3 (m, 5H, aromatic), 7.20 (dd,J=16, 12 Hz, 1H, olefinic), 6.60 (d, J=16 Hz, 1 H, olefinic), 6.38 (d,J=16 Hz, 1 H, olefinic), 6.25 (d, J=12 Hz, 1H, olefinic), 6.15 (d, J=16Hz, 1 H, olefinic), 5.80 (s. 1H, olefinic), 2.48 (s. 3H, CH₃l, 2.05 (t.J=5Hz, 2H, CH₂), 1.79 (s, 3H, CH₃), 1.70-1.40 (m, 4H, CH₂—CH₂), 1.00 (s,6H, 2×CH₃)

[0202] 9-phenyl-9-cis RA: TLC Rf 0.23 (13% ethyl acetate in Benzene)

Example 11 Synthesis of 4-hydroxy-9-cis-retinoic Acid

[0203] To a solution of 9-cis-retinoic acid (51 mg, 0.17 mmole) in1.4-dioxane (2 ml) was added SeO₂ (19 mg, 0.17 mmole) at 60° C. Thesolution was allowed to stir at that temperature for 3 hours. Thereaction mixture was then filtered through a silica bed. The filtratewas concentrated and the residue subjected to flash columnchromatography (silica, 75% ether in petroleum ether) to afford4-OH-9-cis-retinoic acid (21 mg., 40% yield), which is characterized asfollows: Oil; TLC Rf=0.25 (silica, 75% ether in petroleum ether); ¹HNMR(400 MHz, CDCl₃) δ 7.08 (dd, J=16, 12 Hz, 1H, olefinic); 6.64 (d, J=16Hz, 1H, olefinic), 6.21 (d, J=16 HZ, 1H, olefinic), 6.20 (d, J=16 Hz,1H, olefinic); 6.04 (d, J=12 Hz, olefinic), 5.79 (s, 1H, olefinic), 4.02(t, J=5 Hz, 1H, CH—O ), 2.18 (s, 3H, CH₃), 2.02 (s, 3H, CH₃), 1.82 (s,3H, CH₃), 2.0-1.6 (m, 4H, CH₂—CH₂), 1.05, 1.03 (2×s, 2×3H, 2×CH₃).

Example 12 Synthesis of 4-keto-9-cis-retinoic Acid

[0204] To a solution of 4-hydroxy-9-cis-retinoic acid (16 mg, 0.05mmole) in CH₂Cl₂ (1.5 ml) was added Dess-Martin reagent [see Dess andMartin in J. Org. Chem. 48:4155 (1983)] (42 mg, 0.1 mmole) in oneportion at 25° C. After stirring for 5 minutes, the mixture was dilutedwith 10 ml of ether and to this was added saturated aqueous NaHCO₃ (5ml) containing Na₂SO₃ (55 mg). The mixture was stirred for 20 minutes todissolve the solid and the layers separated. The ether layer was washedwith H₂O (2×5 ml), brine (5 ml) and dried (MgSO₄). The solvent wasrecovered under reduced pressure and residue was subjected to flashcolumn chromatography (silica, 60% ether in Hexane) to give4-keto-9-cis-retinoic acid (14 mg. 90%), characterized as follows: TLCrf=0.6 (silica, 80% ether in hexane); ¹HNMR (400 mHz, CDCl₃) δ 7.05 (dd,J=16, 12 Hz, 1H, olefinic), 6.82 (d, J=16 Hz, 1 H, olefinic), 6.32 (d,J=16 Hz, 1H, olefinic), 6.30 (d, J=16 Hz, 1H, olefinic), 6.20 (d, J=12Hz, 1H, olefinic), 5.80 (s, 1H, olefinic), 2.5 (t, J=7 Hz, 2H, CH₂—CO),2.31 (s, 3H, CH₃), 2.01 (s, 3H, CH₃), 1.9 (s, 3H, CH₃), 1.89 (m, 2H,CH₂), 1.20 (s, 6H, 2×CH₃)

Example 13 In Vitro Evaluation of 9-phenyl-9cis-retinoic Acid,4-hydroxy-9-cis-retinoic Acid and 4-keto-9-cis-retinoic Acid

[0205] The potency and efficacy of the compounds described in Examples10, 11 and 12 were determined (as described in Example 1—under theheading “Cotransfection Assay in CV-1 Cells”. The results are presentedin Table 4: TABLE 4 9-cis-retinoic 9-phenyl-9-cis- 4-hydroxy-9-cis-4-keto-9-cis- acid retinoic acid retinoic acid retinoic acid PotencyPotency Potency Potency Receptor (nM) Efficacy (nM) Efficacy (nM)Efficacy (nM) Efficacy RXRα 88 170% 210   76% 1700 161% 520 104% RXPβ 61106% 44   88% 650 143% 1300 105% RXRγ 360 137% 290   77% 1700 115% 1100133% RARα 99  94% >10,000 <2% 380  65% 200  50% RAPβ 22  97% 880   39%160  71% 26  67% RARγ 43 108% 250   59% 180  81% 55 107%

[0206] While the invention has been described in detail with referenceto certain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

1 2 71 amino acids amino acid unknown protein internal 1 Cys Xaa Xaa CysXaa Xaa Asp Xaa Ala Xaa Gly Xaa Tyr Xaa Xaa Xaa 1 5 10 15 Xaa Cys XaaXaa Cys Lys Xaa Phe Phe Xaa Arg Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa XaaXaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35 40 45 Xaa Xaa XaaLys Xaa Xaa Arg Xaa Xaa Cys Xaa Xaa Cys Arg Xaa Xaa 50 55 60 Lys Cys XaaXaa Xaa Gly Met 65 70 16 base pairs nucleic acid both both DNA (genomic)2 TCAGGTCATG ACCTGA 16

That which is claimed is:
 1. A composition in unit dosage form for oraladministration consisting essentially of as an active ingredient acompound selected from the group consisting of 9-cis retinoic acid,pharmaceutically acceptable salts thereof, pharmaceutically acceptablehydrolyzable esters thereof and a pharmaceutically acceptable carriersuitable for oral administration.
 2. A composition in unit dosage formfor oral administration consisting of as an active ingredient a compoundselected from the group consisting of 9-cis retinoic acid,pharmaceutically acceptable salts thereof, pharmaceutically acceptablehydrolyzable esters thereof and a pharmaceutically acceptable carriersuitable for oral administration.